1H, 13C, 15N Backbone Assignment of the Minimally Tied Trefoil Knot, MTTSA , a 23s rRNA SPOUT Methyltransferase

Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from Staphylococcus aureus , MTT _SA (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 3 ₁ knot. Here, we report the non-proline backbone assignments with 98.8% completion using TROSY-based NMR experiments on uniformly labeled ² H/ ¹³ C/ ¹⁵ N-labeled protein. Complementary HBHA(CO)NH experiments on ¹³ C/ ¹⁵ N-labeled protein were completed for Hα/Hβ sidechain assignments. Both backbone and sidechain assignments were deposited in the Biological Magnetic Resonance Bank (BMRB ID: 53391). Of 164 assigned residues, TALOS-N predictions provided 88.4% unambiguous assignments based on the H _N , H _α , C _α , C _β and N chemical shifts. In addition, the predicted secondary structure based on torsion angels and the ΔδC _α -ΔδC _β profile are in agreement with the crystal structures.